Abstract-In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca 2ϩ ] i , which may in turn affect vascular tone. One such mechanism is the extrusion of Ca 2ϩ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca 2ϩ ] i or Ca 2ϩ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS. Key Words: transgenic mice Ⅲ blood pressure Ⅲ nitric oxide synthase Ⅲ intracellular calcium Ⅲ myogenic tone H ypertension is a polygenic disease responsive to environmental factors. Although more than 50 genes have been implicated in the regulation of blood pressure, 1 the complexity of hypertension has been additionally revealed by studies suggesting that insertion or deletion of single genes can lead to either reduced or elevated blood pressure. 2 One hallmark of hypertension is increased peripheral resistance attributable to both structural and functional changes in resistance arteries. 3 On a functional level, peripheral resistance is a balance between vasodilator and vasoconstrictor mechanisms of vascular smooth muscle cells (SMCs), in turn dependent on the intracellular calcium concentration ([Ca 2ϩ ] i ) and the Ca 2ϩ sensitivity of the contractile apparatus. Homeostasis of Ca 2ϩ involves several distinct mechanisms, including the extrusion of Ca 2ϩ by plasma membrane calcium ATPase (PMCA). These high-affinity calmodulin-responsive Ca 2ϩ efflux pumps are P-type transport proteins encoded for by a family of 4 genes (PMCA1 through PMCA4). Both PMCA1 and PMCA4 are ubiquitously expressed, whereas PMCA2 and PMCA3 show cell-specific patterns of expression. 4 From these 4 genes, more than 20 distinct isoforms of PMCA ...

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r 2/2 ), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r 2/2 mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.Type 2 diabetes (T2D) is associated with a number of risk factors that contribute to an increased risk of atherothrombotic events, including hypertension, dyslipidemia, obesity, and chronic inflammation, as well as endothelial and platelet dysfunction (1). Platelets are small, versatile, anucleate cells in the circulation that play critical roles in both early and late stages of atherothrombosis, contributing also to cell-based thrombin generation and blood coagulation (2). Subjects with T2D exhibit a prothrombotic state, including increased production of coagulation factors; decreased production of fibrinolytic factors; and a propensity to platelet activation, aggregation, and adhesion (1,3,4). Compounding the latter, subjects with T2D show reduced sensitivity to antiplatelet drugs, such as aspirin and clopidogrel (5,6), and manifest a higher incidence of cardiovascular events (1,6,7). Although the currently available antidiabetic agents have been effective at lowering blood glucose levels and preventing microvascular disease, until the recent EMPA-REG study (8), it had been exceedingly difficult to demonstrate the beneficial effects of normalizing blood glucose

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ObjectiveTo understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR).BackgroundLifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia.MethodsMale 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI.ResultsThis short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

. Calcineurinindependent regulation of plasma membrane Ca 2ϩ ATPase-4 in the vascular smooth muscle cell cycle. Am J Physiol Cell Physiol 285: C88-C95, 2003; 10.1152/ajpcell.00518.2002.-Calcineurin mediates repression of plasma membrane Ca 2ϩ -ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45 Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca 2ϩ concentrations ([Ca 2ϩ ]i) increased twofold, and real-time RT-PCR and Western blot revealed a ϳ40% decrease in PMCA4 expression levels from G 0 to G1/S in the cell cycle, where PMCA4 constituted ϳ20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca 2ϩ -independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca 2ϩ ]i through alternate targets. /calmodulin-mediated signals act at multiple points in the cell cycle, including the G 0 /G 1 transition, the initiation of S-phase, and the initiation and completion of M phase (reviewed in Refs. 4,31,38,and 44 ] i ) needed for G 1 -to-S transitions in rat vascular smooth muscle cells (VSMC) (22, 23).We previously showed that the cell cycle-associated repression of PMCA1 expression during G 0 to G 1 /S progression in rat VSMC is mediated by the c-Myb transcription factor (1). However, the mechanism(s) underlying the cell cycle-associated repression of PMCA4 had not been elucidated. Guerini et al. (18) demonstrated in mouse neurons that PMCA4 expression can be repressed by a calcineurin-dependent pathway. Given this result, we hypothesized that G 1 /Sassociated repression of PMCA4 expression in VSMC may also be mediated by calcineurin. To explore these mechanisms in cell culture, we generated a clonal, immortalized mouse VSMC line (MOVAS). Immunostaining for smooth muscle-specific proteins such as SM22␣, calponin, smooth muscle-specific ␣-actin and desmin, as well as SM22␣ promoter-driven enhanced green fluorescent protein (EGFP) expression, confirm the lineage and phenotype of these cells. 45 Ca efflux assays and fura 2-based ratiometric Ca 2ϩ imaging reveal regulated Ca 2ϩ efflux and [Ca 2ϩ ] i at the G 1 /S transition point. Western blot and real time RT-PCR reveal cell cycle-regulated repression of mouse PMCA1 and PMCA4. Drugs in...

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Abstract-Inhibiting activity of the c-Myb transcription factor attenuates G 1 to S phase cell cycle transitions in vascular smooth muscle cells (SMCs) in vitro. To determine the effects of arterial SMC-specific expression of a dominantnegative c-Myb molecule (Myb-Engrailed) on vascular remodeling in vivo, we performed carotid artery wiredenudation in 2 independent lines of binary transgenic mice with SM22␣ promoter-defined Doxycycline-suppressible expression of Myb-Engrailed. Adult mice with arterial SMC-specific expression of Myb-Engrailed were overtly normal in appearance and did not display any changes in cardiovascular structure or physiology. However, bromodeoxyuridinedefined arterial SMC proliferation, neointima formation, medial hyperplasia, and arterial remodeling were markedly decreased in mice expressing arterial SMC-restricted Myb-Engrailed after arterial injury. These data suggest that c-Myb activity in arterial SMCs is not essential for arterial structure or function during development, but is involved in the proliferation of arterial SMCs as occurs in vascular pathology, and that the expression of a dominant-negative c-Myb can dramatically reduce adverse arterial remodeling in an in vivo model of restenosis. As such, this model represents a novel tissue-specific strategy for the potential gene therapy of diseases characterized by arterial SMC proliferation.

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